A LINEAR-REGRESSION METHOD FOR THE STUDY OF THE COOMASSIE BRILLIANT BLUE PROTEIN ASSAY

The interactions of Coomassie brilliant blue G-250 (CBB) with bovine s erum albumin (BSA) and gamma-globulin at low pH are investigated by a spectrophotometric method. It is considered that the binding of CBB to protein is because of the weak interactions (ionic, van der Waals, hy drogen bonding, and hydrophobic). The solution equilibria involving th e binding of three dye species (blue, green, and red) to protein are t reated in the same way as Ringbom model used in the treatment of compl exation in analytical chemistry. Based on this treatment, the formatio n of an isosbestic point in the absorption spectra of CBB-BSA mixtures is discussed, two mathematical models for the description of the CBB protein assay are developed. The first model is a nonlinear equation w hich is rigorous in theory but unreliable in use because of its optimi zation procedure. The second model based on an approximation is a line ar equation, it allows to estimate apparent binding constant, maximum binding number, and molar absorptivity of bound dye from assay data by a linear regression method. The results of the linear regression oper ations are reasonable and in agreement with experimental findings. Fac tors which influence the sensitivity of the CBB protein assay are stud ied using this method. Ionic strength and acidity are found to have si gnificant effect on the binding of CBB to protein. (C) 1997 Elsevier S cience B.V.